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Molecular Characterization of Protozoan Parasites and Ehrlichia in Domestic Animals from Umkhanyakude District of Kwazulu-Natal

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Molecular Characterization of Protozoan Parasites and Ehrlichia in Domestic Animals from Umkhanyakude District of Kwazulu-Natal Molecular characterization of protozoan parasites and Ehrlichia in domestic animals from uMkhanyakude district of KwaZulu-Natal LS Mofokeng orcid.org 0000-0002-0274-1828 Dissertation submitted in fulfilment of the requirements for the degree Master of Science in Environmental Sciences at the North-West University Supervisor: Prof MMO Thekisoe Co-supervisor: Prof NJ Smit Graduation May 2019 29933870 ACKNOWLEDGEMENTS Even though a completed dissertation carries a single name of the student, the process that leads to its achievement is always accomplished in combination with the work of other dedicated people. I wish to acknowledge my sincere appreciation and gratitude to certain people for their invaluable contribution to the study. Professor Oriel M.M. Thekisoe, supervisor of my dissertation, for the patient guidance, encouragement and advice he has provided throughout my time as his student. I have been extremely lucky to have a supervisor who cared so much about my work, and who responded to my questions and queries so promptly. Without his help and encouragement this dissertation would not have been written (or ever finished!). I would also like to thank Professor N.J. Smit, the co-supervisor for his support during my study. Special thanks also go to Dr. Oriel M. Taioe for his assistance on the phylogenetic analysis and for his comments on the manuscript. He taught me how to work and think to the best of my ability. I am indebted to Dr. E. Onyiche for his many helpful suggestions and comments on the statistical analysis. Thanks are also due to Mr Dennis Komape for his assistance during the development of the maps. My colleagues in Molecular parasitology and zoonosis group, especially Dr. N.I. Molefe, Malitaba Mlangeni, Bridget Makhahlela, Clara-lee van Wyk, Anna Seetsi, Siphamandla Lamula and Setjhaba Mohlakoana are acknowledged for their comments and assistance during the development of this dissertation - Thank you very much. I wish to express my wholehearted thanks to my parents, Mofokeng Tokelo and Mofokeng Moliehi. I could never have accomplished this dissertation without their love, support, and understanding. They raised and taught me to study hard and to give priority in my life to the quest for knowledge. I also wish to thank my siblings, Maipato, Tsietsi and Mamokete for doing their best to understand a brother who had to be confined to his study and for their words of encouragement. i This work would not have been possible without the financial support of the DST-NRF innovation master’s scholarship. Last but certainly not least, I acknowledge the generous cooperation of local farmers who participated in this study and the kind cooperation of the veterinarians who helped during the collection of the blood samples. “GOD Thank you for giving me the strength and encouragement especially during all the challenging moments in completing this dissertation. I am truly grateful for your exceptional love and grace during this entire journey”. ii ABSTRACT Protozoan and ehrlichial diseases are a major threat to domestic animals in tropical and sub-tropical regions of Africa. Economically important animal diseases in sub-Saharan Africa include theileriosis, babesiosis, trypanosomosis, hepatozoonosis, toxoplasmosis, besnoitiosis and ehrlichiosis. These diseases have a considerable impact on the country’s economic security and impact negatively on poor communities who are depended on livestock production as their source of income and nutritional needs, and as labour for fieldwork and transport. As such, it is documented that the occurrence of protozoan parasites in South African domestic animals hinders the development of livestock industry, which contributes for up to 49% of the agricultural yield. It is important to keep up to date data on occurrence of these diseases using modern molecular diagnostic techniques. Therefore, this study was aimed at improving the current knowledge about the occurrence and genetic diversity of protozoan parasites and Ehrlichia in domestic animals from north eastern KwaZulu-Natal (KZN). A total of 208 blood samples collected from apparently healthy domestic animals (cattle, dogs, goats and sheep) in three different municipalities of uMkhanyakude district, (KZN) were screened using genus and species-specific PCR techniques for the detection of Besnoitia besnoiti, Theileria spp., Babesia spp., Hepatozoon canis, Trypanosoma spp., Toxoplasma gondii, and Ehrlichia canis species-specific genes. The PCR amplicons were sequenced for detected species confirmation and phylogenetic analysis. The maximum likelihood trees were constructed to evaluate genetic diversity between protozoan parasites and Ehrlichia sequences of randomly selected isolates. Overall infection rates of T. ovis in sheep, B. bigemina, B. bovis in cattle and Trypanosoma spp. in cattle, T. gondii in cattle and Ehrlichia canis in dogs were 3 (30%), 33 (30.3%), 24 (22.2%), 20 (18.35%), 5 (4.58%), 20 (40.8%), respectively. The co-infection of two pathogens were detected in 4 (3.7%) for B. bovis and B. bigemina. The generated nucleotide sequences were confirmed to correspond with GenBank strains of respective PCR positive species. Analysis of phylograms constructed with RAP-1, B1, 18S and 16S sequences of B. bovis, T. gondii and E. canis indicated a close relationship between isolates detected in this study and GenBank strains. On the other hand, a tree constructed with SpeI-AvaI restriction fragment sequences revealed a high degree of polymorphism among the B. bigemina isolates investigated in this study. Taken together, the results of the current iii study indicated that protozoan parasites are prevalent in domestic animals from uMkhanyakude district of KZN province. A large scale epidemiological study covering the rest of the district municipalities in KZN province is needed, in order to provide a clearer picture of the prevalence of these protozoan and ehrlichial pathogens in domestic animals. Ultimately, this prevalence data will contribute in formulation of control strategies against diseases caused by these pathogens. Key Terms: Ehrlichia canis, Toxoplasma gondii, Trypanosoma, Babesia, Theileria, Besnoitia, uMkhanyakude district, Phylogeny, Domestic animals. iv RESEARCH OUTPUTS Lehlohonolo S Mofokeng, Moeti o Taioe, Nico J Smit, Oriel M.M Thekisoe. Molecular characterization of haemoparasites infecting livestock in uMkhanyakude district. 16-18 September 2018. 47th Annual PARSA conference (page 25). Tshepise Forever Resort, Limpopo, South Africa. v TABLE OF CONTENTS 1.1. Background ...................................................................................................... 1 1.1.1. Trypanosoma...................................................................................................... 2 1.1.1.1 Classification of trypanosomes ........................................................................... 2 1.1.1.2 General life cycle of trypanosomes ..................................................................... 2 1.1.1.3 Characterization of trypanosomes ...................................................................... 3 1.1.1.4 Epidemiology of African animal trypanosomosis ................................................. 4 1.1.1.5 Pathogenesis and clinical signs of trypanosomosis ............................................ 5 1.1.1.6 History of nagana in KwaZulu-Natal province ..................................................... 5 1.1.2. Apicomplexans ................................................................................................... 6 1.1.2.1. Piroplasmids ....................................................................................................... 7 1.1.2.2. Clinical courses and life cycles of piroplasmida .................................................. 8 1.1.2.2.1. Theileria.............................................................................................................. 8 1.1.2.2.1.2. January disease (Zimbabwe theileriosis) ............................................................ 9 1.1.2.2.2. Life cycle of Theileria .......................................................................................... 9 1.1.2.2.3. Babesia ............................................................................................................ 10 1.1.2.2.3.1. Canine babesiosis ............................................................................................ 11 1.1.2.2.3.2. Bovine babesiosis ............................................................................................. 11 1.1.2.2.3.3. Ovine babesiosis .............................................................................................. 12 1.1.2.3. Epidemiology of piroplasmosis ......................................................................... 13 1.1.2.4. Coccidians ........................................................................................................ 14 1.1.2.4.3. Besnotia besnoiti .............................................................................................. 14 vi 1.1.2.4.3.1. Transmission and clinical signs of bovine besnoitiosis ...................................... 15 1.1.2.4.3.2. Life cycle of Besnoitia ..................................................................................... 15 1.1.2.4.3.3. Epidemiology of besnoitiosis ........................................................................... 16 1.1.2.4.4. Toxoplasma gondii ........................................................................................... 17 1.1.2.4.4.1.
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